Identification of the miaB Gene, Involved in Methylthiolation of Isopentenylated A37 Derivatives in the tRNA of Salmonella typhimurium andEscherichia coli

ABSTRACT The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is deficient in the methylthio group of the modified nucleosideN6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A37). By sequencing, we found that the Tn10dCm of this strain had been inserted into thef474 (yleA) open reading frame, which is located close to the nag locus in both S. typhimurium and Escherichia coli. By complementation of the miaB2508::Tn10dCm mutation with a minimal subcloned f474 fragment, we showed thatf474 could be identified as the miaB gene, which is transcribed in the counterclockwise direction on the bacterial chromosome. Transcriptional studies revealed two promoters upstream ofmiaB in E. coli and S. typhimurium. A Rho-independent terminator was identified downstream of themiaB gene, at which the majority (96%) of themiaB transcripts terminate in E. coli, showing that the miaB gene is part of a monocistronic operon. A highly conserved motif with three cysteine residues was present in MiaB. This motif resembles iron-binding sites in other proteins. Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms2i(o)6A37 formation.